Antimicrobial susceptibility tests are among the most important tests performed in the clinical microbiology laboratory. Administration of antibiotics to patients whose infections are caused by bacteria resistant to those antibiotics seriously compromises and delays effective therapy. Resistance to antibiotics is mediated by several mechanisms. In the case of antibiotics containing a beta-lactam ring structure, such as the penicillins, cephalosporins, carbapenems, cephamycins, and monobactams, resistance is conferred by beta-lactamases, which hydrolyze the beta-lactam ring, thereby inactivating the antibiotic.
The genes encoding the beta-lactamases may be constitutively expressed by plasmids carried by the bacterial host, or may reside in the bacterial chromosome. Class I beta-lactamases are of the latter type and are induced following exposure to a beta-lactam antibiotic. These enzymes are produced in finite but very low amounts in the uninduced state. Class I beta-lactamases include the cephalosporinases, which inactivate all beta-lactam antibiotics including penicillins, cephalosporins, cephamycins, and monobactams. Virtually all strains of Citrobacter freundii, Enterobacter spp., Moraanella moraanii, Proteus vulgaris, Providencia spp., Pseudomonas aeruginosa, and Serratia spp. contain class 1 beta-lactamases.
It is known that class I beta-lactamases are induced to varying degrees by different beta-lactam antibiotics, as summarized by Sanders, et al., Eur. J. Clin. Microbiol., 6: 435 (1987). This means that some antibiotics such as cefoxitin and imipenem are excellent inducers, resulting in production of high levels of beta-lactamases. Others, however, such as ticarcillin, ceftazidime, mezlocillin, piperacillin, carbenicillin and ceftriaxone are not particularly good inducers of enzyme. Since the level of enzyme is too low to quantitate in the repressed (uninduced) state, assays in the prior art have adopted a format in which beta-lactamase is first induced with an inducing antibiotic, and then the organism is challenged with the indicator antibiotic.
One such assay has been described by Sanders, et al., Antimicrobial Agents and Chemotherapy, 15:792 (1979) for an agar dilution technique. In this assay a Petrie dish is overlayed with agar containing the pregrown target organism. A first disk impregnated with the inducing antibiotic is placed on the agar surface. A second disk impregnated with the indicator antibiotic is placed about 10 millimeters from the first disk. The Petrie dish is incubated for some 18-24 hours.
As the bacterial lawn grows up, the antibiotics diffuse from the disks into the surrounding agar. If the organism is susceptible to the indicator antibiotic, a completely round clear zone of inhibition is observed. If the organism is induced, the zone of inhibition is flattened on the edge between the two antibiotic disks. The explanation for the result is that there is a diffusion race in which the embedded incipient colonies of the target organism closer to the inducing antibiotic become resistant through induction of the protective beta-lactamase before the indicator antibiotic has time to reach them. FIG. 1 of the Example hereinbelow illustrates this test.
Sanders, et al., J. Infect. Dis., 154: 792 (1986) described an alternate method for detection of beta-lactam resistant organisms, in which the target strain is first grown up in the presence of an inducing antibiotic. The cells are collected, lysed, and beta-lactamase activity is measured in the supernatant.
Mett, et al., Eur. J. Clin. Microbiol., 7: 669 (1988) described a relatively rapid test in which bacteria carrying a gene for inducible beta-lactamase expression are inoculated into microtiter plates for determination of the minimum inhibitory concentration. After 4 hours of incubation, a chromogenic beta-lactamase substrate is added and changes in color are monitored over the next three hours of incubation. A similar approach was reported by Jarlov, et al., Acta Pathol. Microbiol. Immunol. Scand., 94: 415 (1986).
There have been several studies in which combinations of inducing and noninducing beta-lactam antibiotics have been utilized. See, for example, Pederson, et al., J. Antimicrob. Chemother., 19: 101 (1987). The object of making these combinations is to assess a synergistic potential of the combinations against particularly intractable bacterial strains.
In general, the current methods of determining beta-lactam susceptibility and resistance, namely, agar dilution, broth microdilution, Kirby-Bauer, all fail to detect drug resistance in certain gram-negative bacteria, as discussed by Jorgensen, Chemiotherapia, 4: 7 (1985). The known methods all require a pregrowth step in which the strain is grown in broth or on a plate under conditions in which the organism is exposed only to the inducing antibiotic. This step is followed by a challenge in the presence of an indicator antibiotic or direct assay of enzymatic activity. These approaches require pure culture inoculation and growth, and involve up to 24 hours incubation.